It is well known that isolated small peptides and proteins are usually only poorly immunogenic. Toxic adjuvants, like the well-known Freunds' complete adjuvant, are widely used to stimulate immune responses against subunit vaccines in animals, but many cannot be used in humans because of their toxic side-effects. The ideal situation would be to avoid completely the use of external adjuvants, but this typically results in poor immune responses. However, the (human) immune system generates robust immune responses against pathogens displaying repeated antigenic structures across a surface, e.g. that of a virus [Zinkernagel, R., Science, (1996), 171, 173-178].
Liposomes have received a great deal of attention over the past 30 years as carriers for pharmaceutical products, including drugs, nucleic acids, and biopharmaceuticals, and applications of liposomes as delivery vehicles for antigens, nucleic acids and drugs are well known. The properties of liposomes can be altered by coupling peptides or proteins to their surface in order to target specific receptors; creating systems known as proteoliposomes. Peptides and proteins have also been incorporated into liposomes for the purpose of generating immune responses [Leserman, L., J Liposome Res, (2004), 14, 175-189; Frisch, B., et al., Methods Enzymol., (2003), 373, 51-73]. The conjugation of peptides to lipids facilitates their insertion into liposomes, with the lipid anchored in the bilayer membrane, thus allowing recognition of the peptide by antibodies at the surface of the liposome. One of the drawbacks of liposomes as a general delivery vehicle is their instability in vivo, due to fast elimination from the blood and capture by the cells of the reticuloendothelial system [Torchilin, V. P., Nat. Rev. Drug Discov., (2005), 4, 145-160].
The potential advantages of using virus-like particles, composed of natural or genetically modified viruses and chimeras, including phages, or natural or genetically modified viral components, such as capsid proteins, surface proteins and glycoproteins, or fragments of these, in vaccine design have been recognized for some time [Felnerova, D., et al., Curr Opin Biotechnol, (2004), 15, 518-529; Garcea, R. L., et al., Curr Opin Biotechnol, (2004), 15, 513-517; Doan, L. X., et al., Rev Med Virol, (2005), 15, 75-88]. The production of such virus-like particles makes use of the natural viral processes of self-assembly. The natural self-assembling core structures of many viruses can be exploited using recombinant DNA technology to display one or more antigens on the surface of these particles. These virus-like particles are not accessible by chemical synthesis due to their large size and structural complexity. Patent applications WO 98/014564 and WO 00/035479 refer to “synthetic virus-like particles”, although the particles referred to therein are based on natural or genetically modified virus particles, or components thereof, made using recombinant DNA and cell-based methods, not materials produced by chemical synthesis. WO 00/32227 describes the use of core particles of natural or non-natural origin to which antigens are attached in an ordered and repetitive fashion, exemplified by the use of recombinant Sinbis virus.
Considerable efforts have also been made to design self-assembling peptides and proteins for nanotechnological applications. Nanoscale morphologies have been produced based on designed amphiphilic peptides [Löwik, D. W. P. M., et al., Chem. Soc. Rev., (2004), 33, 234-245], including those having β-strand, β-sheet and α-helical secondary structures [Rajagopal, K., et al., Curr. Opin. Struct. Biol., (2004), 14, 480-486; Tu, R. S., et al., Adv. Drug Deliv. Revs., (2004), 56, 1537-1563]. Further examples of the use of lipopeptides to prepare nanostructured composite materials are found in the work of Stupp and co-workers [Behanna, H. A., et al., J. Am. Chem. Soc., (2005), 127, 1193-1200]. A peptide amphiphile was shown earlier to self-assemble into nano-fibres [Hartgerink, J. D., et al., Science, (2001), 294, 1684-1688].
One of the main problems in designing effective vaccines based on synthetic antigens has been their poor immunogenicity. Relatively small synthetic molecules tend to be poorly immunogenic. One approach to overcome this poor immunogenicity is to covalently conjugate the synthetic antigen to a carrier, such as a protein like tetanus toxin or keyhole limpet hemocyanin (KLH) [Herrington, D. A., et al., Nature, (1987), 328, 257-259]. The conjugate, however, must still be administered to an animal together with an adjuvant (e.g. alum or Freunds' adjuvant) in order to elicit a strong immune response. A number of other methods have been described for producing multi-epitope constructs incorporating B-cell and T-cell epitopes (reviewed in [Jackson, D. C., et al., Vaccine, (1999), 18, 355-361]).
Synthetic bacterial lipopeptide analogs have received wide attention in vaccine research, both for their adjuvant effects and as carriers for peptide antigens [Ghielmetti, M., et al., Immunobiology, (2005), 210, 211-215]. Lipids and lipopeptides are known to be capable of adjuvanting otherwise weak peptide immunogens [Jung, G., et al., Angew. Chem. Int. Ed., (1985), 10, 872; Martinon, F., et al., J. Immunol., (1992), 149, 3416]. Many lipopeptide constructs have been reported, in which a lipid with known adjuvant effects has been coupled to a peptide to generate self-adjuvanting vaccine candidates. Particularly well studied are tripalmitoyl-S-glyceryl cysteine (N-palmitoyl-S-(2,3-bis-(O-palmitoyloxy)-propyl)-cysteinyl- or Pam3Cys) and dipalmitoyl-S-glyceryl cysteine (2,3-bis-(O-palmitoyloxy)-propyl)-cysteinyl- or Pam2Cys) [Ghielmetti, M., et al., Immunobiology, (2005), 210, 211-215]. These lipid moieties are found in lipoprotein components of the inner and outer membranes of gram-negative bacteria. Synthetic lipopeptides carrying these or related di-acylated or tri-acylated S-glyceryl cysteine residues at the N-terminus have been shown to be specific ligands of Toll-like receptors [Reutter, F., et al., J. Pept. Res., (2005), 65, 375-383; Buwitt-Beckmann, U., et al., Eur. J. Immunol., (2005), 35, 1-8]. Moreover, the conjugation of peptide antigens to Pam3Cys or Pam2Cys has been applied in the design of self-adjuvanting synthetic vaccine candidates (Bessler, W. G., et al., Int. J. Immunopharmac., (1998), 19, 547-550; Loleit, M., et al., Biol. Chem. Hoppe-Seyler, (1990), 371, 967-975; Muller, C. P., et al., Clin. Exp. Immunol., (1989), 78, 499-504]. Patent application WO 98/07752 describes the use for drug targeting purposes of lipopeptides, wherein the peptide portion may be a collagen-like sequence capable of inducing triple helical structures.
A number of reviews on coiled-coil design have appeared recently [Woolfson, D. N., Adv. Prot. Chem., (2005), 70, 79-112], including a volume of Advances in Protein Chemistry devoted to coiled-coils, collagen and elastomers [Parry, D. A. D., et al., Advancs in Protein Chemistry, (2005), 70]. Many natural viruses and microbes contain coiled-coil peptide sequences within their own surface proteins (e.g. hemagglutinin of influenza virus, or gp41 of human immunodeficiency virus-1 (HIV-1), or the F-glycoprotein of respiratory syncytial virus (RSV)).